Lee Anderson has resigned as deputy Conservative chairman to rebel over the government’s flagship Rwanda bill.
Another deputy party chairman, Brendan Clarke-Smith, also resigned ahead of voting against the government.
In a joint letter to the prime minister, the pair said they supported the legislation but wanted to make sure it was “watertight”.
They were among 60 Tory MPs who backed an amendment which rebels said was aimed at toughening up the bill.
The legislation seeks to revive the government’s plan to send some asylum seekers to Rwanda and deter people from crossing the Channel in small boats.
In their resignation letter, the pair said they had previously argued that “safeguards” were needed to ensure the legislation was “watertight”.
“It is therefore important in terms of credibility that we are consistent with this,” they added.
The Tory MPs said they had supported rebel amendments to the Rwanda bill “not because we are against the legislation, but because like everyone else we want it to work”.
Both represent so-called “red wall” seats previously held by Labour and have been outspoken on the need to tackle illegal immigration.
With strong support from the right of the party, there had been speculation Prime Minister Rishi Sunak would choose not to sack them as they held party not government positions.
But in their letter, they acknowledged their “important roles” meant they were bound by collective responsibility – so are expected to vote with the government or resign.
Amendments tabled by Robert Jenrick – who resigned as immigration minister last year over the Rwanda legislation – and veteran Conservative Sir Bill Cash were among those voted on by MPs.
Sir Bill’s proposal, which aimed to prevent any international law being used to block someone being removed to Rwanda, was defeated 529 votes to 68.
Senior Tories including former Home Secretary Suella Braverman and former Prime Minister Liz Truss were among those voting in favour.
Mr Jenrick’s amendment was also defeated, by 525 votes to 58.
Under his proposal, people could still appeal their removal to Rwanda, but only after they had been deported.
The bigger test for the government is expected to come on Wednesday, when some of the rebels could vote against the entire bill.
If around 30 Tory MPs joined opposition parties in voting against the legislation it could be defeated.
At least four Tories – including Mr Jenrick and Mrs Braverman – have publicly said they are prepared to vote against the bill if it is not improved, with reports that more could join them.